PROJECT SUMMARY Among vertebrates, several species are capable of regenerating portions of their limbs upon amputation, including: newts, salamanders, and larval frogs (which can regenerate their entire limbs), many teleosts including zebrafish (which can regenerate portions of their fins), and some rodents and primates (which can regenerate their digit tips). All of these examples of appendage regeneration occur by epimorphic regeneration, a process which relies upon the recruitment, proliferation, and differentiation of a population of progenitor cells, termed the blastema. Despite decades of study, the molecular process by which the blastema gives rise to a perfectly formed regenerate remains enigmatic. Intriguingly, there are many morphological similarities between the processes of epimorphic regeneration and embryonic limb development, supporting the hypothesis that appendage regeneration may be regulated by the same molecular pathways that govern limb development. To address this hypothesis, we utilize the mouse digit tip, as it is a robust experimental model for epimorphic regeneration, in a human-relevant mammalian system with established genetic tools. We aim to interrogate the role of defined genetic molecular pathways necessary for embryonic limb patterning, during mouse digit tip regeneration. In Aim 1, we determine if the genes En1 or Lmx1b are necessary for dorsoventral patterning of the regenerating mouse digit tip, as they both are in the developing limb. In Aim 2, we address the role of retinoic acid signaling in proximodistal patterning during digit tip regeneration, and use a transcriptome approach to more broadly define any proximodistal patterning pathways in common with limb development. Collectively, this proposed research will advance our limited understanding of epimorphic regeneration, specifically, how the blastema is molecularly patterned to give rise to a properly formed regenerate in three axes. Understanding whether this process utilizes defined embryonic pathways, or novel regeneration-specific pathways, will clarify our broad understanding of epimorphic regeneration, and will set the field on a trajectory to dramatically change translational regenerative medicine.